Cell membranes are made of lipids capable of forming a barrier between aqueous compartments. They consist primarily of a continuous double or bilayer plate of lipid molecules associated with various membrane proteins. The phospholipids, sphingolipids, and glycolipids make up the three major classes of membrane forming lipid molecules. These lipids are amphipathic (amphiphilic) molecules in that they have a hydrophilic (polar) head and a hydrophobic (non-polar) tail. In the aqueous environment of cells, the polar head groups face toward the water while their hydrophobic tail groups interact with each other to create a lamellar bilayer, and to a lessor extent other aggregate structures depending on the lipid composition and conditions. For example, membrane lipids can form a variety of different shapes including spheres (vesicles), rods (tubes) and lamellae (plates) depending on lipid and water content, and temperature. These shapes represent basic units that interact to form two- and three-dimensional lattice matrix structures classified as lamellar phase (e.g., bilayer plate, closed sphere), hexagonal phase (e.g., rod), or cubic phase (e.g., spheres, rods or lamellae connected by aqueous channels) (Lindblom, et al., Biochimica et Biophysica Acta (1989) 988:221-256). A cross section of a typical cell membrane bilayer (lamellar phase) of phospholipid can be viewed as having a hydrophobic core region of about 30 Angstroms (Å) with two interfacial regions of about 15Å each (White, et al., Curr. Struc. Biol. (1994) 4:79-86).
Proteins can associate with cell membranes in different ways. Integral membrane proteins contain at least one component that is embedded within the lipid bilayer. The non-polar segments of these integral membrane proteins, which embed in the lipid bilayer perpendicular to the surface of the membrane, may consist of a hydrophobic region of the polypeptide, a covalently attached fatty acid chain or other types of lipid chains. Peripheral membrane proteins normally associate with the lipid bilayer through non-covalent interactions with these integral membrane proteins. Additionally, some peripheral membrane proteins are located entirely in the aqueous phase, associated with the membrane through a covalently attached fatty acid or lipid chain. The co-translational attachment of a fatty acid chain such as myristic acid to the amino-terminal glycine of a protein through an amide linkage results in localization of the protein to the cytoplasmic face of cellular membranes. Prenyl groups and palmitic acid groups are attached post-translationally via thioether linkages to cysteine residues and also result in localization of proteins to the membrane. These types of covalent attachments are important for function in a wide variety of cell signaling proteins, like the heterotrimetric G proteins (James, et al., Biochemistry (1990) 29(11):2623-2634; Morello, et al., Biochem. Cell Biol. (1996) 74(4):449-457; Mumby, S. M., Curr. Opin. Cell. Biol. (1997) 9(2):148-154; Resh, M. D., Cell Signal (1996) 8(6):403-412; and Boutin, J. A., Cell Signal (1997) 9(1):15-35). Glycosylphosphatidylinositol anchors, found at the C-terminus of soluble proteins, result in the attachment of these proteins to the cell surface membrane (Turner, A. J., Essays Biochem. (1994) 28:113-127).
The two major classes of known integral membrane proteins are those that insert α-helices into the lipid bilayer, and those proteins that form pores in the lipid bilayer by β-barrel strands (Montal, et al., Curr. Opin. Stuc. Biol. (1996) 6:499-510; Grigorieff,et al., J. Mol. Biol. (1996) 259:393-42; and Weiss, et al., J. Mol. Biol. (1992) 227:493-509). Single membrane spanning proteins, or single-pass membrane proteins, generally have a hydrophobic region that anchors that sequence in the lipid bilayer via an α-helix configuration. Multiple membrane spanning proteins, or multi-pass membrane proteins, result from the polypeptide chain passing back and forth across the lipid bilayer and typically employ cc-helix and/or β-barrel structured membrane anchors.
Examples of membrane proteins include membrane-associated receptors, transporter proteins, enzymes, and immunogens. For instance, cell membrane-associated receptors represent a dynamic collection of membrane proteins of particular therapeutic importance. Four basic superfamilies are recognized: the enzyme-linked receptors, the fibronectin-like receptors, the seven transmembrane receptors, and the ion channel receptors. Enzyme-linked receptors represent single-pass membrane proteins, with the basic structure consisting of a single polypeptide traversing the plasma lamella once via an α-helix anchor domain. The extracellular domain of enzyme-linked receptors binds hormone/ligand, while the carboxyl-terminal domain contains a catalytic site that promotes signal transduction via hormone/ligand binding and receptor aggregation.
The fibronectin-like receptors have the same general structure as the enzyme-linked receptors except that no specific catalytic site is represented in the cytoplasmic domain. Class 1 fibronectin-like receptors contain two modified extracellular domains formed from two seven stranded β-sheets that join at right angles to a ligand-binding pocket. The class 2 fibronectin-like receptors have a slightly different structure in that they form repeats of five-stranded β-sheets that extend over the hormone like fingers. The class 1 and 2 receptors contain a conserved proline-rich cytosolic juxtamembrane region that constituatively binds soluble tyrosine kinases, which is activated by ligand/hormone-binding and receptor aggregation.
The seven-transmembrane receptors, also called G-protein coupled receptors, serpentine receptors, or heptahelical receptors, represent the largest and most diverse family of membrane receptors identified to date. These receptors mediate sensory and endocrine related signal transduction pathways and are multi-pass membrane proteins having α-helical anchor regions that transverse the membrane seven times. The transmembrane spanning regions for some of these proteins form a small ligand/hormone-binding pocket, while larger binding sites are formed through extended amino terminal regions. Seven-transmembrane receptors also contain one or more intracellular loops that bind and activate G-proteins, which act as second messengers in cells.
The ion channel receptors are represented by the ligand- and voltage-gated channel membrane protein receptors. Ligand-gated ion channels are formed by pentamers of homologous subunits. Each subunit contributes an α-helix toward forming the wall of the channel. Ligand/hormone binding appears to occur between the subunits. The typical voltage-gated channel receptors are homotetramers, with each subunit having six transmembrane α-helices.
Different techniques have been used to study membrane proteins and/or exploit them for therapeutic purposes, diagnostics, and drug screening assays and the like. However, unlike non-membrane proteins, the biggest obstacle in working with membrane proteins is the poor solubility of their hydrophobic polypeptide chains, the difficulty in folding membrane proteins from unfolded polypeptide chains and the difficulty in overexpressing and isolating them in environment suitable for quantitative analyses (Huang, et al., J. Biol. Chem. (1981) 256:3802-3809; and Liao, et al., J. Biol. Chem. (1983) 258:9949-9955). For example, unfolding and folding whole transmembrane proteins is difficult since they are insoluble in the lipid bilayer in the unfolded form, as well as in the aqueous phase in both their folded and unfolded forms, because of their highly hydrophobic character (Haltia, et al., Biochimica et Biophysica Acta (1995) 1241:295-322). This feature of membrane proteins is particularly problematic when attempting to synthesize, label or otherwise manipulate them chemically in a cell free environment. Nevertheless, individual transmembrane segments of membrane proteins have been chemically synthesized via solid phase chemistry, followed by subsequent insertion into membranes and spontaneous assembly of native-like structures with biological activity (Popot, et al., Biochemistry (1990) 29:4031-4037; and Grove, et al., Methods Enzymol. (1992) 207:510-525). To date, however, solid phase synthesis has been limited to synthesis of only a few short transmembrane peptide segments, since membrane proteins are recalcitrant to standard chemical synthesis techniques.
Establishing access to membrane proteins with site-specific chemical modifications is crucial both for the analysis of structure-function relationships of membrane proteins and for drug discovery. The most important techniques currently employed to achieve this goal are the synthesis of small membrane-spanning peptide fragments of these proteins (Grove, et al., Methods Enzymology (1992) 207:510-525; and MacKenzie, et al., Science (1997) 276:131-133), chemical modification of existing or engineered cysteine residues (Oh, et al., Science (1996) 273:810-812), and in vitro suppression mutagenesis to incorporate unnatural amino acids (Cload, et al., Chemistry and Biology (1996) 3:1033-1038; and Turcatti, et al., J. Biol. Chem. (1996) 271:19991-19998). None of these techniques provides general access to totally synthetic or semi-synthetic membrane proteins containing chemically modified amino acid side-chains, or their production in a quantity sufficient for most biophysical techniques. Additionally, such techniques do not permit modular synthesis and reassembly of membrane-incorporated transmembrane polypeptide segments or domains.